PROJECT SUMMARY Anxiety is a pervasive public health issue. Throughout the course of their lifetimes, one third of Americans will experience an anxiety disorder ranging from mild to incapacitating, and the prevalence of anxiety is rising. One suspected reason for this trend is that modern society promotes social isolation due to career demands and reliance on technology. Benzodiazepines (BZs) are widely used to provide rapid, effective anxiety relief by enhancing inhibitory signaling at synaptic GABAA receptors (GABAARs) throughout the brain. However, BZs cause side effects including dependence and sedation, and bind only to GABAARs containing gamma subunits. Social isolation downregulates gamma subunits in the rodent brain, reducing BZ efficacy. One brain region crucial for both anxiety and social behaviors is the lateral septum (LS). Inhibitory GABA-releasing (GABAergic) neurons predominate in the LS, but whether activity in the LS induces or relieves anxiety is controversial due to its broad interconnectivity with other regions. Because of this complexity, previous studies have not identified the precise functional changes in LS circuitry that occur during experiences like social isolation. Since ideal treatments for anxiety are unavailable, it is necessary to understand LS circuitry to design better drugs that selectively target anxiety circuits and not the entire brain. Thus, the objective of this proposal is to elucidate some of the specific GABAergic circuitry responsible for isolation-induced anxiety. An underexplored connection exists from the LS to the nucleus accumbens (NAc), a region that is thought to assign positive reward value to prosocial interaction. Pathophysiology in the NAc contributes to mental illnesses that are co-morbid with anxiety disorders, and social isolation decreases excitability in the NAc medial shell. In line with this, inhibiting the NAc caudal medial shell induces fearful behaviors. Therefore, the central hypothesis of this proposal is that social isolation activates an inhibitory GABAergic LS-to-NAc circuit, which encodes social reward deprivation and associated anxiety behaviors. To test this, two aims are proposed. Aim #1 endeavors to investigate isolation-induced physiological changes in NAc-projecting LS neurons. The hypothesis is that excitatory glutamatergic input to LS-to-NAc neurons is enhanced during social isolation, inhibitory input is reduced, and these neurons are less sensitive to BZ inhibition. Aim #2 proposes to define how LS-to-NAc neurons influence expression of social or anxiety behaviors. The hypothesis is that LS-to-NAc neurons directly inhibit the NAc during isolation, which decreases exploratory and prosocial behaviors. To accomplish these aims, the proposed experiments will use simultaneous two-color optogenetics (SToCO), chemogenetics (DREADDs), and behavioral assays in mice. Successful fulfillment of these aims will have immediate impact by revealing novel functions of a circuit from a region critical for social and anxiety behaviors (the LS) to a region critical for reward evaluation (the NAc). By uncovering the circuit and synaptic changes that link social isolation to anxiety, the proposed studies will allow for the development of superior therapeutics.